The present invention relates generally to a novel method for the preparation of nucleoside analogues and their precursors and more particularly to a method of preparing a nucleoside analogue by the use of specific enzymes to stereoselectively produce dioxolane nucleoside analogues or their precursors.
An important class of pharmacological agents relate to 3xe2x80x2-oxa-substituted 2xe2x80x2,3xe2x80x2-dideoxynucleoside analogues (xe2x80x9cdioxolane nucleoside analoguesxe2x80x9d). These compounds include 9-(xcex2-D-2-hydroxymethyl-1,3-dioxolan-4-yl)-2-aminopurine (xcex2-D-DAPD); 9-(xcex2-D-2-hydroxymethyl-1,3-dioxolan-4-yl)-guanine (xcex2-D-DXG); 1-(xcex2-L-2-hydroxymethyl-1,3-dioxolan-4-yl)-thymine (Dioxolane-T); and 1-(xcex2-L-2-hydroxymethyl-1,3-dioxolan-4-yl)-cytidine (xcex2-L-OddC) which have known antiviral and antitumor activity.
As shown in the following dioxolane structure, dioxolanes have two chiral centers corresponding to the substituted carbons 2 and 4 of the dioxolane ring (C2 and C4 respectively). Thus each compound can exist as four different stereoisomers depending on the position of both substituents with respect to the dioxolane ring. 
The stersoisomers of a dioxolane nucleoside analogue are represented by the following diagrams where the letter B represents a purine or pyrimidine base or an analogue or derivative of a purine or pyrimidine base as defined herewith. 
For the purpose of consistency, the same stereochemical designation will be used even when the hydroxymethyl moiety or the base moiety (B) is replaced with another substituent group.
Chiral synthetic methods have improved over the past several years with respect to synthetic techniques that result in single stereoisomer compounds. However, there is a present need to find novel synthetic methods which can be widely used to form a particular stereoisomer with greater efficiency and purity.
For example, for many years a person of ordinary skill in the art could use enzymes to separate enantiomers of dioxolane compounds. However, there is still a need in the art to produce a dioxolane nucleoside analogue using a step of separating an anomeric mixture of certain dioxclane precursors to produce an end product with greater efficiency and purity.
Because stereochemically pure dioxolane nucleosides are an important class of compounds due to their known antiviral activity and anticancer activity, there is a need for other inexpensive and efficient stereoselective methods for their preparation. The present invention satisfies this and other needs.
The present invention provides a novel process for making dioxolane nucleoside analogues with a high degree of steric purity, greater efficiency and higher yields.
Specifically, the present invention provides a process for making dioxolane nucleoside analogues with a high degree of steric purity which includes the use of certain hydrolytic enzymes for separating xcex2 and xcex1 anomers from an anomeric mixture represented by the following formula A or formula B: 
wherein R1 is selected from the group consisting of C1-6 alkyl and C6-15 aryl; Bz is Benzoyl.
The process involves the step of hydrolyzing the mixture of compounds represented by formula A and/or formula B with an enzyme selected from the group consisting of Protease N (Bacillus subtilis protease), Alcalase(copyright) (Subtilisin Carlsberg protease), Savinase(copyright) (Bacillus lentus subtilisin protease), ChiroCLEC(trademark)-BL (Bacillus licheniformis Subtilisin protease), PS-30 (Pseudomonas cepacia lipase), and ChiroCLEC(trademark)-PC (Pseudomonas cepacia lipase). The process stereoselectively hydrolyses predominantly one anomer to form a product where R1 of formula A and formula B is replaced with H. The other anomer remains substantially unhydrolysed. The process also comprises separating the hydrolyzed product from unhydrolysed starting material.
According to one embodiment of the invention, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 97% xcex2-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 98% xcex2-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 98.5% xcex2-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 98.8% xcex2-anomer.
According to one embodiment of the invention, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 97% xcex1-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 98% xcex1-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 98.5% xcex1-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 98.8% xcex1-anomer.
In one embodiment, the xcex2-anomer is the predominant product. In another embodiment, the xcex1-anomer is the predominant product. In yet another embodiment, the xcex2-L-enantiomer is the predominant product. In an additional embodiment, the xcex2-D-enantiomer is the predominant product. In yet another embodiment, the xcex1-L-enantiomer is the predominant product. In an additional embodiment, the xcex1-D-enantiomer is the predominant product.
In one embodiment, the invention is a process for stereoselectively preparing a dioxolane nucleoside analogue by separating xcex2 and xcex1-nomers from an anomeric mixture represented by formula A or formula B according to one of the above embodiments. The process further includes the step of stereoselectively replacing the functional group at the C4 position (COOR1) with a purinyl or pyrimidinyl or analogue or derivative selected from the group consisting of: 
In this embodiment, R2, R9 and R11 are independently selected from the group consisting of hydrogen, C1-6 alkyl, C1-6 acyl and R8C(O) wherein R8 is hydrogen or C1-6 alkyl. Additionally, R3, R4 and R10 are each independently selected from the group consisting of hydrogen, C1-6 alkyl, bromine, chlorine, fluorine, iodine and CF3; and R5, R6 and R7 are each independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, amino, hydroxyl and C3-6 cycloalkylamino. The process results in the production of a stereochemical isomer of the dioxolane nucleoside analogue.
According to one embodiment, the process further includes the step of stereoselectively replacing the functional group at the C4 position (COOR1) with a purinyl or pyrimidinyl or derivative selected from the group consisting of: 
In this embodiment, R2 is selected from the group consisting of hydrogen, C1-6 alkyl, C1-6 acyl and R8C(O) wherein R8 is hydrogen or C1-6 alkyl. Additionally, R3 and R4 are each independently selected from the group consisting of hydrogen, C1-6 alkyl, bromine, chlorine, fluorine, iodine and CF3; and R5, R6 and R7 are each independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, amino, hydroxyl and C3-6 cycloalkylamino. The process results in the production of a stereochemical isomer of a dioxolane nucleoside analogue.
In another embodiment, the process further includes the step of stereoselectively replacing the functional group at the C4 position (COOR1) with a pyrimidinyl or analogue or derivative selected from the group consisting of: 
In this embodiment, R9 and R11 are independently selected from the group consisting of hydrogen, C1-6 alkyl, C1-6 acyl and R8C(O). Additionally, R10 is selected from the group consisting of hydrogen, C1-6 alkyl, bromine, chlorine, fluorine, iodine and CF3. The process results in the production of a stereochemical isomer of a dioxolane nucleoside analogue.
In another embodiment, the process comprises stereoselectively preparing a dioxolane nucleoside analogue by separating xcex2 and xcex1 anomers from an anomeric mixture represented by formula A or formula B according to one of the above embodiments and further comprises stereoselectively replacing the functional group at the C4 position (COOR1) with a moiety selected from the group consisting of: 
In another embodiment of the present invention, the process comprises making a dioxolane nucleoside analogue by separating a compound according to formula A or formula B. According to this embodiment, the process includes stereoselectively replacing the R group with a 9-purinyl or 1-pyrimidinyl moiety or analogue or derivative thereof by acylating the second mixture to produce an acylated second mixture. This embodiment also includes the step of glycosylating the acetylated second mixture with a purine or pyrimidine base or analogue or derivative thereof and a Lewis Acid to produce a dioxolane nucleoside analogue.
The present invention involves a high yield process of separating xcex2 and xcex1 anomers from an anomeric mixture of dioxolane nucleoside analogue precursors which provides higher yield and greater efficiency. In one embodiment, this method is used in the production of dioxolane nucleoside analogues having a high degree of anomeric purity at lower cost. Additionally, another aspect of the present invention involves synthesizing starting material having a higher degree of anomeric purity.
The present invention provides a process of preparing dioxolane nucleoside analogues having a predominant xcex2-L-configuration using enzymes, namely hydrolases. The procedure improves overall yield and has relatively few steps, thereby improving overall efficiency. The process involves the following steps.
A mixture of anomers represented by formula A or formula B is obtained as described herein in Scheme 1. 
In the above formula, R1 is selected from the group consisting of H, C1-6 alkyl and C6-15 aryl and Bz is Benzoyl. The mixture is hydrolyzed with an enzyme selected from the group consisting of Alcalase(copyright) (Subtilisin Carlsberg protease, Novo Nordisk), Savinase(copyright) (Bacillus lentus substilisin protease, Novo Nordisk), ChiroCLEC(trademark)-BL (Bacillus licheniformis Subtilisin protease, Altus Biologics, Inc.), PS-30 (Pseudomonas cepacia lipase, Amano), Protease N (Bacillus subtilis protease, Amano) and ChiroCLEC(trademark)-PC (Pseudomonas cepacia lipase, Altus Biologics, Inc.). The hydrolyzing step stereoselectively hydrolyzes the xcex1-anomer of the mixture of either formula A or formula B. The result is an unhydrolyzed xcex2-anomer. The xcex1-anomer can be separated easily from the xcex2-anomer. If an anomeric mixture of the compound of formula A is selected, the result is the production of the compound of formula C and formula D: 
If an anomeric mixture of the compound of formula B is selected, the result is the production of the compound of formula E and formula F: 
The mixture (C)/(D) or (E)/(F) is then subjected to oxidative decarboxylation which replaces the R1 group with an acyl moiety. It is then glycosylated with a purine or pyrimidine base or analogue or derivative thereof in the presence of a Lewis Acid. The final step produces a dioxolane nucleoside analogue in the xcex2-L configuration for the mixture (C)/(D) and a dioxolane nucleoside analogue in the xcex2-D configuration for the mixture (E)/(F).
At the outset, the following definitions have been provided as reference. Except as specifically stated otherwise, the definitions below shall determine the meaning throughout the specification.
xe2x80x9cNucleosidexe2x80x9d is defined as any compound which consists of a purine or pyrimidine base, linked to a pentose sugar.
xe2x80x9cDioxolane nucleoside analoguexe2x80x9d is defined as any compound containing a dioxolane ring as defined hereinafter linked to a purine or pyrimidine base or analogue or derivative thereof. A xe2x80x9cdioxolane ringxe2x80x9d is any substituted or unsubstituted five member monocyclic ring that has an oxygen in the 1 and 3 positions of the ring as illustrated below: 
xe2x80x9cPurine or pyrimidine basexe2x80x9d is defined as the naturally occurring purine or pyrimidine bases adenine, guanine, cytosine, thymine and uracil. A purine or pyrimidine that is a moiety is a purinyl or pyrimidinyl, respectively.
xe2x80x9cAlkylxe2x80x9d is defined as a substituted or unsubstituted, saturated or unsaturated, straight chain, branched chain or carbocyclic moiety, wherein the straight chain, branched chain or carbocyclic moiety can be optionally interrupted by one or more heteroatoms (such as oxygen, nitrogen or sulfur). A substituted alkyl is substituted with a halogen (F, Cl, Br, I), hydroxyl, amino or C6-20 aryl.
xe2x80x9cArylxe2x80x9d is defined as a carbocyclic moiety which can be optionally substituted or interrupted by one heteroatom (such as oxygen, nitrogen or sulfur) and containing at least one benzenoid-type ring (such as phenyl and naphthyl).
xe2x80x9cCarbocyclic moietyxe2x80x9d is defined as a substituted or unsubstituted, saturated or unsaturated, C3-6 cycloalkyl wherein a substituted cycloalkyl is substituted with a C1-6 alkyl, halogen (i.e. F. Cl, Br, I), amino, carbonyl or NO2.
A xe2x80x9cderivativexe2x80x9d of a purine or pyrimidine base refers to one of the following structures: 
wherein one or more of the pyrimidine H are substituted with substituents that are known in the art. In the above illustration, the bonds represented by a broken line are optional and are present only in cases which require the bond to complete the valence of the ring atom. Substitutents bound to the ring members by a single bond include but are not limited to halogen such as F, Cl, Br, I; an akyl such as lower akyls; aryl; cyano carbamoyl; amino including primary, secondary and tertiary amino; and hydroxyl groups. Substituents bound to the carbon ring atoms by a double bond include but are not limited to a xe2x95x90O to form a carbonyl moiety in the ring. It is understood that when the ring is aromatic, some of the substitutions may form tautomers. The definition shall include such tautomers.
xe2x80x9cAnaloguexe2x80x9d of a purine or pyrimidine base refers to any derivative of purine or pyrimidine bases that is further modified by substituting one or more carbon in the ring structure with a nitrogen.
xe2x80x9cStereoselective enzymesxe2x80x9d are defined as enzymes which participate as catalysts in reactions that selectively yield one specific stereoisomer over other stereoisomers.
xe2x80x9cAnomeric purityxe2x80x9d is defined as the amount of a particular anomer of a compound divided by the total amount of all anomers of that compound present in the mixture multiplied by 100%.
xe2x80x9cAlkoxyxe2x80x9d is defined as an alkyl group, wherein the alkyl group is covalently bonded to an adjacent element through an oxygen atom (such as methoxy and ethoxy).
xe2x80x9cAlkoxycarbonylxe2x80x9d, is defined as an alkoxy group attached to the adjacent group of a carbonyl.
xe2x80x9cAcylxe2x80x9d is defined as a radical derived from a carboxylic acid, substituted (by a halogen, C6-20 aryl or C1-6 alkyl) or unsubstituted by replacement of the xe2x80x94OH group. Like the acid to which it is related, an acyl radical may be aliphatic or aromatic, substituted (by halogen, C1-6 alkoxyalkyl, nitro or O2) or unsubstituted, and whatever the structure of the rest of the molecule may be, the properties of the functional group remain essentially the same (such as acetyl, propionyl, isobutanoyl, pivaloyl, hexanoyl, trifluoroacetyl, chloroacetyl and cyclohexanoyl).
xe2x80x9cAlkoxyalkylxe2x80x9d is defined as an alkoxy group attached to the adjacent group by an alkyl group (such as methoxymethyl).
xe2x80x9cAcyloxyxe2x80x9d is defined as an acyl group attached to the adjacent group by an oxygen atom.
xe2x80x9cOxoxe2x80x9d is defined as a xe2x95x90O substituent bonded to a carbon atom.
xe2x80x9cHydroxy protecting groupxe2x80x9d is well known in the field of organic chemistry. Such protecting groups may be found in T. Greene, Protective Groups in Organic Synthesis, (John Wiley and Sons, 1981). Examples of hydroxy protecting groups include but are not limited to benzyl, benzoyl, substituted benzoyl, acetyl and substituted acetyl.
As noted above, one embodiment of the present invention is a process for separating xcex2 and xcex1 anomers from an anomeric mixture represented by the following formula A or formula B: 
wherein R1 is selected from the group consisting of C1-6 alkyl and C6-15 aryl; Bz is Benzoyl.
Another embodiment of the present invention is a process for separating xcex2 and xcex1 anomers from an anomeric mixture represented by the following formula Axe2x80x2 or formula Bxe2x80x2: 
wherein R1 is selected from the group consisting of C1-6 alkyl and C6-15 aryl; W is a hydroxy protecting group.
In one embodiment, the process stereoselectively hydrolyses predominantly the xcex1-anomer to form a product where R1 of formula A and formula B is replaced with H. The xcex2-anomer remains substantially unhydrolyzed. The process also comprises separating the hydrolyzed product from unhydrolyzed starting material.
The process of making a xcex2-L dioxolane nucleoside analogue begins with the preparation of starting materials. Scheme 1 depicts the manufacture of a mixture that includes formula A or B. 
A benzoyloxyacetaldehyde (formula 1A) is reacted with 1,3-dioxolane-4-carboxylic acid-2,2-dimethyl-methyl ester (formula 1B) in approximately equimolar proportions. The dioxolane of formula 1B has a chiral center at-the C4 carbon. The reaction occurs in a toluene solvent. The mixture is heated to 58xc2x0 C. The catalyst, PTSA, is added. The mixture is heated to a temperature between 64-67xc2x0 C. A vacuum is applied at 70 kPa, and the reaction proceeds for 40 minutes. Traces of solvent are then removed by high vacuum. The catalyst is removed by filtration using a 1:1 ratio of Hexane:EtOAc as an eluent. In one embodiment, the preferred filter is a silica gel pad. The resulting product is a crude oil containing a mixture of the compounds of formula 1C and 1D wherein the ratio is 2:1 of (1C:1D), respectively.
It can be appreciated by a person of skill in the art that the reaction conditions can be adjusted to optimize the purity of the stereoisomers. In one embodiment of the present invention, the reaction of the compound of formula 1A with the compound of formula 1B is done in the presence of catalyst in an amount between about 1.0 wt % and 10.0 wt % of the starting material. In another embodiment the amount of catalyst is between about 2.5 wt % and about 5.5 wt % of the starting materials. In yet another embodiment, the amount of catalyst is between about 3.0 wt % and about 5.0 wt %. In still another embodiment, the amount of catalyst is between about 3.5%. and about 5.5%. In another embodiment, the amount of catalyst is between about 2.5 wt % and about 7.5 wt %. In another embodiment the amount of catalyst is about 5.0 wt %.
In an embodiment of the present invention, the reaction of the compound of formula 1A with the compound of formula 1B is done at a temperature ranging from about 40xc2x0 C. to about 80xc2x0 C. In another embodiment of the present invention, the temperature ranges from about 50xc2x0 C. to about 75xc2x0 C. In still another embodiment, the temperature ranges from about 60xc2x0 C. to about 70xc2x0 C. In an additional embodiment, the temperature ranges from about 65xc2x0 C. to about 79xc2x0 C.
In an embodiment of the present invention, the reaction time between the compound of formula 1A and the compound of formula 1B corresponds to a period ranging from about 30 minutes to about 2 hours. In yet another embodiment, the period ranges from about 30 minutes to about 1 hour. In still another embodiment, the period ranges from about 30 minutes to about 50 minutes.
It will be appreciated by a person of ordinary skill in the art that the C4 carbon is chiral. Because this carbon is not involved in the reaction, the chirality is preserved at that carbon. A starting material can be selected to have a (4S) or (4R) stereochemistry.
According to one embodiment, it is preferable that the resulting product is an anomeric mixture favoring the xcex2-L configuration over the xcex1-L configuration. To achieve such a result, the starting material represented by formula 1B (4S) is selected and shown below: 
The reaction proceeds according to the principles described above. The resulting product, according to one embodiment, will have an anomeric purity of the xcex2-L anomer over the xcex1-L anomer of greater than 55%, preferably 60% and more preferably 65%.
According to one embodiment, the present invention is a method of separating xcex2-anomers from xcex1-anomers according to the following Scheme 2: 
According to one embodiment, a mixture of anomers is obtained as represented by formula 2A or formula 2B. A mixture represented by formula 2A or formula 2B can be obtained according to the reaction described above or according to any method known in the art.
The reaction is prepared as follows: A portion of the material containing a mixture of compounds represented by formula 2A and formula 2B is weighed into a reaction vessel. According to one embodiment, about 3.7% mmol of the mixture is added to 10 mL of 20% acetonitrile/aqueous buffer. In another embodiment for a preparative scale reaction, about 75.2 mmole of the mixture is added to about 200 ml of 20% acetonitrile/aqueous buffer. The buffer is a phosphate buffer with a pH between 7.0 and 7.5 and preferably 7.2. In another embodiment a 20% aqueous t-butyl methyl ether was used.
The enzyme is selected from the group consisting of Alcalase(copyright) (Subtilisin Carlsberg protease), Savinase(copyright) (Bacillus lentus subtilisin protease), ChiroCLEC(trademark)-BL (Bacillus licheniformis Subtilisin protease), PS-30 (Pseudomonas cepacia lipase), Protease N (Bacillus subtilis protease), and ChiroCLEC(trademark)-PC (Pseudomonas cepacia lipase). These enzymes are commercially available. Particularly, some of the materials can be obtained from the following sources: Savinase(copyright) and Alcalase(copyright) can be obtained from Novo Nordisk. ChiroCLEC(trademark)-BL and ChiroCLEC(trademark) can be obtained from Altus Biologics, Inc. PS-30 and Protease N can be obtained from Amano Pharmaceutical.
The stereospecific enzyme selected is then added to begin the hydrolysis reaction. The enzymatic reaction hydrolyzes primarily the xcex1-anomer by replacing the R1 group of the xcex1-anomer of the compound of formula 2B with H to form the compound of formula 2C. The amount of the enzyme added can be determined according to principles known by any person of ordinary skill in the art. According to another embodiment, about 500 mL was added to begin the reaction. The rate and degree of hydrolysis was monitored by a pH-stat according to principles known in the art. As the compound of formula 2B is hydrolyzed, the pH of the mixture decreases. Thus, the change in pH as monitored by a pH-stat corresponds to the completeness of the reaction.
If the reaction time is allowed to proceed longer than the optimal reaction time, the xcex2-anomer may be converted resulting in lower chemical yield of the final product. If the reaction time is too short, less than optimal amount of the xcex1-anomer is converted resulting in a lower anomeric purity of the remaining unhydrolyzed reactant. According to one embodiment, the reaction is allowed to proceed until 43% completion. It will be appreciated by a person of ordinary skill in the art that the exact degree of completion may change depending upon the reactant used, the enzyme used and other principles known to a person of ordinary skill in the art.
As noted, the ester starting material and the hydrolysed product are separated by increasing the pH of the solution to more than pH 7.0 and in one embodiment below pH 7.5 with sodium bicarbonate and extracting with ethyl acetate (for example, 3xc3x9780 mL). The unhydrolysed starting material is extracted in the ethyl acetate and the hydrolysed product remains in salt form in the aqueous solution. The pH of the solution is then adjusted to pH 2. The hydrolyzed product is further extracted with ethyl acetate (for example, 3xc3x9780 mL). The reactants and the products are dried with MgSO4, filtered and concentrated in-vacuo.
Additionally, the unhydrolysed product can be hydrolysed by procedures known in the art such as reaction with LiOH followed by acidification.
Because of the enzyme selectivity, the anomeric purity of the hydrolyzed and separated xcex1-anomer is considerably high.
According to one embodiment of the invention, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 97% xcex2-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 98% xcex2-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 98.5% xcex2-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 98.8% xcex2-anomer.
According to one embodiment of the invention, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 97% xcex1-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 98% xcex1-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 98.5% xcex1-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 98.8% xcex1-anomer.
In another embodiment, the procedure of Scheme 2 is followed except the anomeric mixture represented by formula 2A and 2B is replaced with an anomeric mixture represented by formula 2D and 2E, respectively. 
According to this embodiment the xcex1-anomer represented by formula 2E is hydrolyzed The result is the separation of the hydrolyzed xcex1-anomer represented by formula 2F from the unhydrolyzed xcex2-anomer represented by formula 2D. 
In another embodiment, the procedure of Scheme 2 is followed except a mixture represented by formula 2A and 2B is replaced with a mixture of four stereoisomers represented by formula 2G. 
According to this embodiment, the xcex1-anomer containing both D and L enantiomers is hydrolyzed. The result is the separation of the hydrolyzed xcex1-anomer containing both D and L enantiomers from the unhydrolyzed xcex2-anomer containing both D and L enantiomers.
After hydrolysis, purification and oxidative decarboxylation, the resulting dioxolane ring can be linked with a purine or pyrimidine base or analogue or derivative. There are several examples known by skilled artisan on how to link a purine or pyrimidine base or analogue or derivative to the dioxolane ring. For example, PCT Publ. No. WO/97/21706 by Mansour et al. describes one method of stereoselectively attaching the purine or pyrimidine base or analogue or derivative to a dioxolane ring. WO/97/21706 is incorporated herein fully by reference.
According to the process disclosed in WO/97/21706 the starting material is an acylated dioxolane ring. The starting material of the procedure disclosed in WO/97/21706 can be obtained by oxidative decarboxylation of a product of Scheme 2 discussed above. Oxidative decarboxylation destroys the stereochemistry of the C4 carbon while preserving the stereochemistry of the C2 carbon.
As noted, the oxidative decarboxylation step occurs after the hydrolysis step of Scheme 2. A compound having the desired stereochemistry on the C2 carbon is selected. For each mmol of compound that is processed, it is dissolved in between about 2.5 and about 4.0 mL of acetonitrile. In another embodiment, between about 3.0 and about 3.5 mL of acetonitrile was added for each mmol of compound. In yet another embodiment, between about 3.3 and about 3.4 mL of acetonitrile was added for each mmol of compound.
For each mmol of compound, between about 0.08 and about 0.12 mL of pyridine was added. In another embodiment, between about 0.09 and about 0.11 mL of pyridine was added for each mmol of compound. In yet another embodiment, approximately 0.1 mL of pyridine was added for each mmol of compound.
To this mixture, between 1.1 and 1.5 mmoles of Pb(OAc)4 was added for each mmol of compound. In another embodiment, between about 1.2 mmoles and about 1.4 mmoles of Pb(OAc)4 is added for each mmol of compound. In yet another embodiment, about 1.3 mmoles of Pb(OAc)4 is added for each mmol of compound.
Thereafter, the mixture was stirred for 18 hours at room temperature. Then, the mixture was poured into a saturated solution of NaHCO3. Between approximately 2.0 and 3.0 mL of NaHCO3 were used for each mmol of compound. In one embodiment, between about 2.5 mL and about 2.7 mL, and more preferably about 2.6 mL of NaHCO3 was used for each mmol of compound. The solution was then stirred for an additional 30 minutes. The organic layer was separated from the aqueous layer by four extractions of ethyl acetate. Extracts were combined, dried on anhydrous Na2SO4 and evaporated under a vacuum. Optionally, the crude can be further purified by chromatography on silica gel using a gradient of 0-15% ethyl acetate in hexane.
In one embodiment of the present invention, the oxidative decarboxylation step is followed by glycosylation. The glycosylation is represented by the following Scheme 3. 
The first step in the glycosylation procedure is to obtain a compound with the desired stereospecificity at the C2 carbon. According to one embodiment, a compound having an S stereochemistry at the C2 carbon, as represented by the compound of formula 3A is preferred. The result is that a higher ratio of the xcex2-L anomer is in the product 3C. According to another embodiment, a compound having an R stereochemistry at the C2 carbon is preferred. The result is a product that has a higher ratio of the xcex2-D anomer in the final product.
The compound of formula 3A is reacted with an iodosilane to produce the compound of formula 3B. In one embodiment, the iodosilane is iodotrimethylsilane.
In another embodiment, the iodosilane is diiodosilane. Important to the reaction is that it occurs at low temperatures. According to one embodiment, the temperature is preferably between 0xc2x0 C. and xe2x88x9278xc2x0 C. prior to glycosylation with silylated pyrimidine or purine base or analogue or derivative thereof. According to another embodiment, the temperature is between 0xc2x0 C. and xe2x88x9214.9xc2x0 C. prior to glycosylation with silylated pyrimidine or purine base or analogue or derivative thereof. According to yet another embodiment, the temperature is between 0xc2x0 C. and xe2x88x9278xc2x0 C. prior to glycosylation with silylated purine base or analogue or derivative selected from the group comprising: 
wherein R5, R6 and R7 are each independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, amino, hydroxyl and C3-6 cycloalkylamino.
According to still another embodiment, the temperature is between 0xc2x0 C. and xe2x88x9278xc2x0 C. prior to glycosylation with silylated purine base or analogue or derivative thereof selected from the group comprising: 
The iodo intermediate represented by formula 3B is then dissolved in dichloromethane and is cooled down to a temperature comparable to the temperature of the reaction vessel.
A purine or pyrimidine base or analogue or derivative thereof is then selected. According to one embodiment, the purine or pyrimidine base or analogue or derivative thereof is selected from the following group: 
wherein R2, R9 and R11 are each independently selected from the group consisting of hydrogen, C1-6 alkyl, C1-6 acyl and R8C(O) wherein R8 is hydrogen or C1-6 alkyl;
R3, R4 and R10 are each independently selected from the group consisting of hydrogen, C1-6 alkyl, bromine, chlorine, fluorine, iodine and CF3; and
R5, R6 and R7 are each independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, amino, hydroxyl and C3-6 cycloalkylamino.
According to one embodiment, the purine or pyrimidine base or derivative is selected from the group consisting of: 
In this embodiment, R2 is selected from the group consisting of hydrogen, C1-6 alkyl, C1-6 acyl and R8C(O) wherein R8 is hydrogen or C1-6 alkyl. Additionally, R3 and R4 are each independently selected from the group consisting of hydrogen, C1-6 alkyl, bromine, chlorine, fluorine, iodine and CF3; and R5, R6 and R7 are each independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, amino, hydroxyl and C3-6 cycloalkylamino.
In another embodiment, the purine or pyrimidine base or analogue or derivative thereof is selected from the group consisting of: 
In this embodiment, R9 and R11 are independently selected from the group consisting of hydrogen, C1-6 alkyl, C1-6 acyl and, R8C(O). Additionally, R10 is selected from the group consisting of hydrogen, C1-6 alkyl, bromine, chlorine, fluorine, iodine and CF3.
The purine or pyrimidine or analogue or derivative thereof is persylated by a sylating agent and ammonium sulphate followed by evaporation of HMDS to form a persylated purine or pyrimidine base or analogue or derivative thereof herein referred to as the persylated base and designated as P in Scheme 3. According to one embodiment, the sylating agent is selected from the group consisting of 1,1,1,3,3,3-hexamethyldisilazane, trimethylsilyl triflate, t-butyldimethylsilyl triflate or trimethylsilyl chloride. In one embodiment, the sylating agent is 1,1,1,3,3,3,-hexamethyldisilazane.
The persylated base P was dissolved in 30 mL of dichloromethane and was added to the iodo intermediate represented by formula 3B. The reaction mixture was maintained at between 0 and 78xc2x0 C. for 1.5 hours then poured onto aqueous sodium bicarbonate and extracted with dichloromethane (2xc3x9725 mL). The organic phase was dried over sodium sulphate to obtain the compound of formula 3C. As used in Scheme 3, the B represents a moiety of the purine or pyrimidine base or analogue or derivative thereof which was persylated in the above step to form P. The compound of formula 3C was removed by filtration and the solvent was evaporated in-vacuo to produce a crude mixture. The product represented by formula 3C has predominantly a 4S configuration at the C4 carbon with an anomeric purity of 80%. When the starting material is a compound represented by formula 3A, the product forms predominantly the xcex2-L enantiomer having an anomeric purity of 80%.
Next, the compound of formula 3C is deprotected to produce the compound of formula 3D. This can be accomplished by dissolving a compound represented by formula 3C in methanol and then adding ammonia or sodium methoxide. The deprotection step can also be done by other methods which are well known by those skilled in the art. The product represented by formula 3D is purified by flash chromatography on silica-gel (5% MEOH in ethylacetate). The deprotection step can also be done by other methods that are well known by a person skilled in the art.
In another embodiment, compounds of Scheme 1 may be prepared by an alternative process which is shown below in Scheme 4. 
Between about 1.0-1.4 eq of sulfuric acid was added in portions to a large excess of water while stirred at a temperature between 0-5xc2x0 C. By way of example and not by limitation, if 9.06 mol of D-Serine represents 1 equivalent of reactant, then between about 9.5-13.3 mol of sulfuric acid is added to 7.3 L of water. In another embodiment, between about 1.1-1.3 eq of sulfuric acid was added to an excess of water. In a further embodiment, 1.2 eq of sulfuric acid was added to an excess of water.
About 1 equivalent of D-Serine was added in one portion under vigorous stirring. Then, between about 1.0 and 1.4 eq. of aqueous sodium nitrite was added dropwise. The temperature was kept between 0-5xc2x0 C. during the addition time (about seven hours). The reaction vessel was stirred overnight at room temperature. The water was removed by vacuum and the residue (D-glyceric acid) co-evaporated with toluene (3xc3x971L). The residue was then stirred with about 6L of an alcohol solvent for about 30 minutes. According to one embodiment, the alcohol is of the formula R1OH wherein R1 is a C1-4 alkyl. According to another embodiment, the alcohol is methanol or ethanol. The resulting solid was removed by filtration. The clear solution was stirred at room temperature for 30-40 hours, the alcohol removed by vacuum to yield a D-glycerate in the form of a yellow viscous syrup. The D-glycerate is then reacted with between about 0.9-1.1 eq of a dialkyl acetal at a temperature of about 85-95xc2x0 C. Examples of suitable dialkyl acetals include benzoyloxyacetaldehyde dialkyl. Examples of suitable alkyls for the dialkyl acetal is methyl and ethyl.
Then, between about 1 wt % and about 10 wt % of PTSA is added. According to another embodiment, about 5 wt % PTSA is added. In another embodiment, about 0.02 eq. of solid PTSA is added. The reaction mixture is kept under vacuum at a temperature between 85-95xc2x0 C. for 2-3 hours. The mixture is then cooled to room temperature, diluted with ethylacetate (250 mL) and poured onto saturated sodium bicarbonate solution (250 mL) under stirring. The organic phase is separated and the aqueous phase concentrated, purified on a silica gel column eluting with 5-10% ethylacetate/hexanes to yield the desired dioxolane as a light yellow oil (about 59%) with xcex2/xcex1 ratio of 2:1 or higher.
Alternatively, the reactants of step 3 of Scheme 4 can be substituted by corresponding reactants of Scheme 1. For example, the D-glycerate represented by Formula 4C is replaced with an 1,3 dioxolane-4-(4R)carboxylic acid-2,2-dimethyl alkyl ester represented by Formula 1B according to one embodiment. Additionally or alternatively, the dialkyl acetal represented by Formula 4D is replaced with a benzoyloxyaldehyde represented by Formula 1A. These substitutions do not require changing the reaction conditions substantially disclosed above for the third step of Scheme 4.
In a further embodiment of the present invention, the starting material of Scheme 4 is L-Serine which produces an end product having an S-configuration at the C4 carbon of the dioxclane ring. Alternatively, the L-glycerate of Step 3 can be replaced with an 1,3 dioxolane-4-(4S)carboxylic acid-2,2-dimethyl alkyl ester to produce an end product having predominantly an S-configuration at the C4 carbon of the resulting dioxolane ring.